Abstract
A nested polymerase chain reaction for detecting Ehrlichia equi in horses and ticks (Ixodes pacificus) was developed. A major second-round PCR product of 928 bp could be readily visualized in ethidium bromide-stained agarose minigels. An internal probe was used to verify the identity of the amplified product by non-radioactive (digoxigenin-based) Southern blotting; additional confirmation was provided by DNA sequence analysis. A dilution study testing the sensitivity of the PCR indicated that DNA derived from ≤ 7.6 but > 3 infected neutrophils was sufficient to generate a PCR signal. The specificity of the PCR was examined using a panel of rickettsiae, of which only E. equi and the closely-related human granulocytotropic ehrlichia produced PCR bands. In an in vivo infection study, E. equi DNA was detected in blood buffy-coat cells of an experimentally-infected horse on days three through 14 post- inoculation. In a separate study, three of six adult I. pacificus that as nymphs had been fed on an experimentally infected horse were found to be PCR- positive for E. equi.
| Original language | English (US) |
|---|---|
| Pages (from-to) | 319-329 |
| Number of pages | 11 |
| Journal | Veterinary Parasitology |
| Volume | 63 |
| Issue number | 3-4 |
| DOIs | |
| State | Published - Jun 1996 |
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Keywords
- Diagnosis Rickettsia
- DNA sequence analysis
- Ehrlichia equi
- Horse
- Ixodes pacificus
- Polymerase chain reaction
- Southern hybridization test
ASJC Scopus subject areas
- Animal Science and Zoology
- Parasitology
- veterinary(all)
Cite this
Nested polymerase chain reaction for detection of Ehrlichia equi genomic DNA in horses and ticks (Ixodes pacificus). / Barlough, Jeffrey E.; Madigan, John E; DeRock, Elfriede; Bigornia, Luisa.
In: Veterinary Parasitology, Vol. 63, No. 3-4, 06.1996, p. 319-329.Research output: Contribution to journal › Article
}
TY - JOUR
T1 - Nested polymerase chain reaction for detection of Ehrlichia equi genomic DNA in horses and ticks (Ixodes pacificus)
AU - Barlough, Jeffrey E.
AU - Madigan, John E
AU - DeRock, Elfriede
AU - Bigornia, Luisa
PY - 1996/6
Y1 - 1996/6
N2 - A nested polymerase chain reaction for detecting Ehrlichia equi in horses and ticks (Ixodes pacificus) was developed. A major second-round PCR product of 928 bp could be readily visualized in ethidium bromide-stained agarose minigels. An internal probe was used to verify the identity of the amplified product by non-radioactive (digoxigenin-based) Southern blotting; additional confirmation was provided by DNA sequence analysis. A dilution study testing the sensitivity of the PCR indicated that DNA derived from ≤ 7.6 but > 3 infected neutrophils was sufficient to generate a PCR signal. The specificity of the PCR was examined using a panel of rickettsiae, of which only E. equi and the closely-related human granulocytotropic ehrlichia produced PCR bands. In an in vivo infection study, E. equi DNA was detected in blood buffy-coat cells of an experimentally-infected horse on days three through 14 post- inoculation. In a separate study, three of six adult I. pacificus that as nymphs had been fed on an experimentally infected horse were found to be PCR- positive for E. equi.
AB - A nested polymerase chain reaction for detecting Ehrlichia equi in horses and ticks (Ixodes pacificus) was developed. A major second-round PCR product of 928 bp could be readily visualized in ethidium bromide-stained agarose minigels. An internal probe was used to verify the identity of the amplified product by non-radioactive (digoxigenin-based) Southern blotting; additional confirmation was provided by DNA sequence analysis. A dilution study testing the sensitivity of the PCR indicated that DNA derived from ≤ 7.6 but > 3 infected neutrophils was sufficient to generate a PCR signal. The specificity of the PCR was examined using a panel of rickettsiae, of which only E. equi and the closely-related human granulocytotropic ehrlichia produced PCR bands. In an in vivo infection study, E. equi DNA was detected in blood buffy-coat cells of an experimentally-infected horse on days three through 14 post- inoculation. In a separate study, three of six adult I. pacificus that as nymphs had been fed on an experimentally infected horse were found to be PCR- positive for E. equi.
KW - Diagnosis Rickettsia
KW - DNA sequence analysis
KW - Ehrlichia equi
KW - Horse
KW - Ixodes pacificus
KW - Polymerase chain reaction
KW - Southern hybridization test
UR - http://www.scopus.com/inward/record.url?scp=0029934164&partnerID=8YFLogxK
UR - http://www.scopus.com/inward/citedby.url?scp=0029934164&partnerID=8YFLogxK
U2 - 10.1016/0304-4017(95)00904-3
DO - 10.1016/0304-4017(95)00904-3
M3 - Article
C2 - 8966998
AN - SCOPUS:0029934164
VL - 63
SP - 319
EP - 329
JO - Veterinary Parasitology: Regional Studies and Reports
JF - Veterinary Parasitology: Regional Studies and Reports
SN - 0304-4017
IS - 3-4
ER -